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Tsukasa, Nobuharu, Takeshi, Hiroki, Takashi, Akira, Nobuo, and Takahiko: Analysis of copy number abnormality (CNA) and loss of heterozygosity (LOH) in the whole genome using single nucleotide polymorphism (SNP) genotyping arrays in tongue squamous cell carcinoma
Original Article

J Korean Assoc Oral Maxillofac Surg 2011;37(6):550-555.

Published online: 10 January 2011

DOI: https://doi.org/10.5125/jkaoms.2011.37.6.550

Analysis of copy number abnormality (CNA) and loss of heterozygosity (LOH) in the whole genome using single nucleotide polymorphism (SNP) genotyping arrays in tongue squamous cell carcinoma

Kuroiwa Tsukasa, Yamamoto Nobuharu, Onda Takeshi, Bessyo Hiroki, Yakushiji Takashi, Katakura Akira, Takano Nobuo, Shibahara Takahiko

Department of Oral and Maxillofacial Surgery, Tokyo Dental College

Shibahara Takahiko Department of Oral and Maxillo-Facial Surgery, Tokyo Dental Colledge 5-2-14-605 Inagekaigan, Mihama-ku, Chiba 261-0005, Japan TEL: +81-43-247-1902 FAX: +81-43-247-1902 E-mail: tkuroiwa@m7.gyao.ne.jp

Copyright © 2011 Korean Association of Oral and Maxillofacial Surgeons

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Chromosomal loss of heterozygosity (LOH) is a common mechanism for the inactivation of tumor suppressor genes in human epithelial cancers. LOH patterns can be generated through allelotyping using polymorphic microsatellite markers; however, owing to the limited number of available microsatellite markers and the requirement for large amounts of DNA, only a modest number of microsatellite markers can be screened. Hybridization to single nucleotide polymorphism (SNP) arrays using Affymetarix GeneChip Mapping 10 K 2.0 Array is an efficient method to detect genome-wide cancer LOH. We determined the presence of LOH in oral SCCs using these arrays. DNA was extracted from tissue samples obtained from 10 patients with tongue SCCs who presented at the Hospital of Tokyo Dental College. We examined the presence of LOH in 3 of the 10 patients using these arrays. At the locus that had LOH, we examined the presence of LOH using microsatellite markers. LOH analysis using Affymetarix GeneChip Mapping 10K Array showed LOH in all patients at the 1q31.1. The LOH regions were detected and demarcated by the copy number 1 with the series of three SNP probes. LOH analysis of 1q31.1 using microsatellite markers (D1S1189, D1S2151, D1S2595) showed LOH in all 10 patients (100). Our data may suggest that a putative tumor suppressor gene is located at the 1q31.1 region. Inactivation of such a gene may play a role in tongue tumorigenesis.

Keywords: Tongue squamous cell carcinoma, Loss of heterozygosity, Copy number abnormality, Single nucleotide polymorphism, tumor suppressor gene

References

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Fig. 1.
DNA labeling, hybridization, washing and staining of the Affymetrix 10K SNP Mapping Array were performed according to the standard Single Primer GeneChip Mapping 10K Assay protocol (Affymetrix). The array was scanned with a GeneChip Scanner 3000, and the scanned array images were processed with GeneChip Operating software. Kuroiwa Tsukasa et al: Analysis of copy number abnormality (CNA) and loss of heterozygosity (LOH) in the whole genome using single nucleotide polymorphism (SNP) genotyping arrays in tongue squamous cell carcinoma. J Korean Assoc Oral Maxillofac Surg 2011
jkaoms-37-550f1.tif
Fig. 2.
Analysis of copy number abnormality (CNA) on 1q31.1 region. Physical positions of each SNP probe are shown at the vertical axis. The copy number of each SNP probe is shown at the horizontal axis. In the region of 1q31.1, genome copy numbers from 0.8 to 1.2 were confirmed from continual SNP probes in all of the 3 cases. Kuroiwa Tsukasa et al: Analysis of copy number abnormality (CNA) and loss of heterozygosity (LOH) in the whole genome using single nucleotide polymorphism (SNP) genotyping arrays in tongue squamous cell carcinoma. J Korean Assoc Oral Maxillofac Surg 2011
jkaoms-37-550f2.tif
Fig. 3.
Analysis of LOH using microsatellite markers (D1S1189, D1S2151, D1S2595). Case numbers are shown at the top and locus symbols on the left. Paired normal (N) and tumor (T) samples for all of 10 patients demonstrating loss of the upper allele (LOH). Kuroiwa Tsukasa et al: Analysis of copy number abnormality (CNA) and loss of heterozygosity (LOH) in the whole genome using single nucleotide polymorphism (SNP) genotyping arrays in tongue squamous cell carcinoma. J Korean Assoc Oral Maxillofac Surg 2011
jkaoms-37-550f3.tif
Table 1.
Summary of clinicopathological features in 10 SCCs
Case Gender Age Region T N Stage Differentiation Mode of invasion pN Final
1 M 41 Tongue 3 2b IV Well 4C − Alive
2 M 58 Tongue 1 1 III Well 3 + Alive
3 M 63 Tongue 1 0 I Well 4C / Alive
4 M 33 Tongue 1 0 I Well 4C + Alive
5 F 60 Tongue 4 2c IV Well 4C + Alive
6 F 63 Tongue 2 1 III Well 1 − Alive
7 M 42 Tongue 1 0 I Poor 4C / Alive
8 M 68 Tongue 1 0 I Well 4C + Died
9 M 56 Tongue 2 0 II Well 4C + Alive
10 M 38 Tongue 1 0 I Poor 4C / Alive

(M, Male; F, Female; pN, pathological Node; Well, well differentiated; Poor, poorly differentiated)

Kuroiwa Tsukasa et al: Analysis of copy number abnormality (CNA) and loss of heterozygosity (LOH) in the whole genome using single nucleotide polymorphism (SNP) genotyping arrays in tongue squamous cell carcinoma. J Korean Assoc Oral Maxillofac Surg 2011

Table 2.
Microsatellite markers used in our LOH study
Markers Locations Size of PCR products (bp) Sequence of primers
D1S1189 1q31.1 341 5'-CTGAACTAACACGGAGAAAC-3' 5'-GTAGACTGTTAAAAGAAGAGC-3' 5'-CTGTATAAAGAGCGCTGTGGG-3'
D1S2151 1q31.1 260
D1S2595 1q31.1 102 5'-GGATGGGGCAGTGAGAAG-3' 5'-GGGCGTTCCAATACTTAGAGG-3' 5'-AGGACACCACAAGTTCCAGG-3'

(LOH: loss of heterozygosity)

Kuroiwa Tsukasa et al: Analysis of copy number abnormality (CNA) and loss of heterozygosity (LOH) in the whole genome using single nucleotide polymorphism (SNP) genotyping arrays in tongue squamous cell carcinoma. J Korean Assoc Oral Maxillofac Surg 2011

Table 3.
Reports of copy number abnormality (CAN) using single nucleotide polymorphism (SNP)
  Reporters Regions
Breast cancer Wang, et al.12 10q23–24, 11q23–24, 13q14, 14q32, 16q23–24, 17p13, 17q11.2–12, 17q21
Bladder cancer Primdahl, et al.14 Hoque, et al.15 Ch6, 8, 9, 11, 17 1p, 2q, 12q, 16p
Prostate cancer Lieberfarb, et al.16 Dumur, et al.17 1p33–34, 3q27, 8p21, 10q23, 15q12, 16q23–24, 17p13 1p31.2, 3p12.1–22.1, 6p25.1–25.3, 8p22–23.2, 10q11.21, 10q22.1, 11q22.1–24.2, 12p13.1, 16p23.1–23.2, 17p13.3, 17q21.31, 19q13.12–13.42
Osteosarcoma Wong, et al.18 6q12–13, 6q14–27
Lung cancer Lindblad-Toh, et al.19 Jänne, et al.20 3p, 4q, 5q, 13q, 15q, 16q, 22q 2p24.1, 2p22.2, 2p15, 2p12, 4q28.2, 6q22.31–23.1, 7q31.2, 7q31.31, 7q36.2, 8p12, 11p14.1, 11p12, 15q26.3, 16q11.1–11.2
Oral SCC-derived cell lines Zhou, et al.23 3p35.3, 11q13.5, 14p14.1–13, 11p, 11q14.3–22.2

(Ch: Chromosome, p: pter, q: qter)

Kuroiwa Tsukasa et al: Analysis of copy number abnormality (CNA) and loss of heterozygosity (LOH) in the whole genome using single nucleotide polymorphism (SNP) genotyping arrays in tongue squamous cell carcinoma. J Korean Assoc Oral Maxillofac Surg 2011

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